Various Raman techniques are known to examine biological and biochemical samples. Linear Raman microscopy consists of irradiating a sample with a strong laser beam and detecting the frequency-modulated detection light emanating from the sample. This technique is, however, too slow to detect processes in the biological and biochemical samples in real-time in a sufficiently precise manner.
In addition, nonlinear Raman techniques are known to solve the problem of the too slow imaging. Among these nonlinear Raman techniques is the observation of coherent anti-Stokes and Stokes scattering (CARS, CSRS). Here, the sample is irradiated collinearly with radiation with two distinct frequencies. As soon as the difference between the two frequencies matches a vibrational resonance frequency in the sample, the sample emits strong Stokes and anti-Stokes radiation which can be detected individually or simultaneously.
A further nonlinear Raman technique which is used for examination under a microscope is stimulated Raman scattering (SRS), in particular ‘Stimulated Raman Gain’ (SRG) or ‘Stimulated Raman Loss’ (SRL). Similar to coherent Stokes or anti-Stokes scattering, the sample is irradiated with radiation with two distinct frequencies. If the sample has a resonance frequency that matches the difference between the two exciting frequencies, energy is transferred from the radiation with higher frequency to the radiation with lower frequency. This change in energy is observed and allows conclusions on the structures and processes in the sample. Here, the observation of the increase in energy for the lower frequency is referred to as SRG and the observation of the respective decrease in energy for the higher frequency is referred to as SRL.
One problem with these techniques is that the exciting radiation and the detection radiation have the same wavelength and polarization. Thus, for the detection signal there results a very strong background signal that makes the detection of the sought-for signal more difficult. One possibility of suppressing the background in the case of SRS is the modulation of the exciting radiation. Another disadvantage of CARS or CSRS is that the image obtained from the detection radiation contains chemically non-specific information as images obtained with CARS or CSRS also comprise non-resonant signals.
A Raman technique which has the advantages but not the disadvantages of the afore-mentioned techniques is the observation of the Raman-induced Kerr effect (RIKES). Here, the sample is again irradiated with radiation of two distinct frequencies, the radiation of one frequency being circularly polarized and the other one being linearly polarized. The RIKES signal then emitted by the sample is polarized orthogonally to the incident radiation of the same frequency. The RIKES signal is fast and chemically specific, however, compared to the other detection signals it is relatively weak.